Differential effects of low and high temperature stress on pollen germination and tube length of mango (Mangifera indica L.) genotypes

Mango flowering is highly sensitive to temperature changes. In this research, the maximum values of pollen germination rate (PGR), pollen tube length (PTL) and their cardinal temperatures (Tmin, Topt and Tmax) were estimated by using quadratic equation and modified bilinear model under the conditions of 14–36 °C. The pollen germination rate in four mango varieties ranged from 29.1% (‘Apple mango’) to 35.5% (‘Renong No. 1’); the length of pollen tube ranged from 51.2 μm (‘Deshehari’) to 56.6 μm (‘Jinhuang’). The cardinal temperatures ranges (Tmin, Topt and Tmax) of pollen germination were 20.3–22.8 °C, 26.7–30.6 °C and 30.4–34.3 °C, respectively; similarly, cardinal temperatures (Tmin, Topt and Tmax) of pollen tube growth were 20.3–21.2 °C, 27.9–32.1 °C and 30.2–34.4 °C respectively. Of those, ‘Renong No. 1’ could maintain relatively high pollen germination rate even at 30 °C, however, ‘Deshehari’ had the narrowest adaptive temperature range. These results were further confirmed by changes of superoxide dismutase, catalase activity and malondialdehyde content. These results showed that mango flowering was highly sensitive to temperature changes and there were significant differences in pollen germination rate and pollen tube length among different varieties. Current research results were of great significance for the introduction of new mango varieties in different ecological regions, the cultivation and management of mango at the flowering stage and the breeding of new mango varieties.


Results
Response of pollen germination and pollen tube growth under temperature stress. Response of pollen germination under temperature stress. The preliminary experiments results on pollen germination and pollen tube growth of different varieties at different temperatures ( Fig. 1) showed that: Overall, after all mango varieties were cultured at 24 ℃ ~ 30 ℃ for 3 h, the pollen germination rate and pollen tube length reached the maximum. Pollen was difficult to germinate below 20 ℃, and then the pollen germination growth rate of most varieties of mango gradually increased with the increase of temperature, reaching the maximum at 28 ℃, with an average germinate rate of 28.3%, and then decreased, the pollen germination rate was less than 10% when the temperature was higher than 32 ℃ ( Fig. 2; Table 1). The growth of pollen tube also indicated similar trend, from the average value of 50.1 μm at 28 ℃ dropped to 34.7 μm at 34 ℃ ( Fig. 3; Table 2).
The quadratic equation (Eq. 1) described the response of pollen germination under temperature stress (R 2 > 0.84). As shown in Fig. 2, the pollen germination rate of mango obviously involved with temperature and variety. The maximum pollen germination rates of the four species were as followed: 'Renong No. 1' 35.5%, 'Dashehari' 32.1%, ' Apple mango' 29.1%, 'Jinhuang' 34.3%, respectively, with the average value of 32.8%. Meanwhile, the basic temperature of different varieties was various. T min ranged from 20.3 ℃ (' Apple mango') to 22.8 ℃ ('Jinhuang'); T opt ranged from 26.7 ℃ (' Apple mango') to 30.6 ℃ ('Renong No. 1'); T max ranged from 30.4 ℃ (' Apple mango') to 34.3 ℃ ('Jinhuang'); the temperature ranged (T max − T min ) from 10.1 ℃ (' Apple mango') to 11.6 ℃ ('Renong No. 1'). According to Table 1, it was found that 'Renong No. 1' pollen had the highest the germination rate and optimum temperature, as well as the widest temperature adaptability, even may maintain a relatively high pollen germination rate at 30 ℃; While the smallest pollen germination rate of ' Apple mango' referred to poor adaptability to the flower period temperature. www.nature.com/scientificreports/   pollen tube had the narrowest range of temperature, and the shortest growth length at the optimum temperature, which largely affected the fruit set and yield of 'Dashehari' . A pollen germination rate of more than 5% was usually considered efficient germination. In terms of the pollen germination rates of four mango varieties, the effective germination temperature of ' Apple mango' was Table 1. Maximum pollen germination percentage and cardinal temperatures for pollen germination of four mango genotypes in response to different temperature stress. *P < 0.05, **P < 0.01.
Pollen germination and culture time in vitro. Pollen culture in vitro for 1 h, 3 h and 5 h at room temperature showed that pollen germination was obvious with the increase of culture time on the culture medium. The pollen germination rate of the four varieties increased sharply within 3 h before culture, and there was no significant difference among the varieties. However, after 5 h of culture, the growth rate of pollen germination of all varieties decreased significantly, and the change of pollen tube length with culture time also showed a similar trend (Tables 3 and 4). On the whole, 'Jinhuang' and 'Renong No. 1' were more tolerant to long-term temperature than 'Dashehari' and ' Apple mango' (Fig. 5).
Cumulative stress response index. In terms of the determination of pollen germination rate and pollen tube length, 28 ℃ temperature stress was used as control; the effects of germination rate under low temperature   50.0% and 57.2% compared to those of under 28 ℃, respectively. It can be seen that there was no significant difference between varieties under low temperature stress and high temperature stress, however, in terms of temperature change rate, high temperature stress had a greater impact on pollen germination rate (Fig. 6a). Similar to pollen germination rate, the pollen tube length of all varieties were higher at 28 ℃ temperature stress, including the highest 'Renong No. 1' , about 53.3 μm, However, under the stress of low temperature and high temperature, the growth of pollen tube degenerated. The pollen length of 'Renong No. 1' , 'Dashehari' , ' Apple mango' and 'Jinhuang' shorted by 55.7%, 53.1%, 53.0% and 57.4% compared to those of under 28 ℃ in the low temperature stress, respectively. Besides, under high temperature stress, the pollen tube length rates of 'Renong No. 1' , 'Dashehari' , ' Apple mango' and 'Jinhuang' were 32.1%, 35.9% and 47.9% and 26.8% compared to those of under 28 ℃, respectively. These results suggested that the inhibition effect of pollen tube growth under low temperature stress was significantly greater than that under high temperature stress (Fig. 6b).   www.nature.com/scientificreports/ In order to detect the pollen sensitivity and tolerance under low temperature and high temperature stress, the CSRI value was calculated ( Response of SOD, POD enzyme activities and MDA content to temperature stress. By measuring SOD and POD enzyme activities in pollen tubes, the response of pollen tubes to low temperature and high temperature stress was studied. The results showed that in the process of pollen germination, low temperature and high temperature treatments caused oxidative stress in pollen tubes, and high temperature had a more significant effect on pollen tubes than low temperature (Figs. 7,8,9).
The activity of protective enzymes was related to the ability of plants to adapt to adversity. Among them, SOD was a key enzyme in plant metabolism, which can maintain the normal metabolism of plants when free radicals were toxic to plants. After low-temperature treatment, the SOD activities of 'Deshehari' , ' Apple mango' and 'Jinhuang' decreased by about 1.7%, 12.8% and 9.2%, respectively, while the SOD activity of 'Renong No. 1' increased by about 4.4%. Under high temperature conditions, the SOD activity of 'Renong No. 1' decreased by 25.1%, 'Deshehari' decreased by 29.9%, ' Apple mango' decreased by 52.9%, and 'Jinhuang' decreased by 45.8% (Fig. 7).

Discussion
With the change of global climate, scientists predicted that the global average temperature will rise by 1.4-5.8 ℃ by 2100 34 , as well as more acute temperature change, resulting in tremendous threat to growth of plant. This study aimed to explore the best mango genotype in different ecological zones by taking the determined pollen parameters as one of the parameters of breeding program due to the similar response trends of pollen germination rate in vitro and pollen tube length under temperature stress. However, because the basic temperature range among mango varieties was relatively narrow, it is necessary to further explore the germplasm resources of mango to determine the tolerance genotype of extreme weather. No matter the extreme climate at present than the trend of global warming in the future, it is necessary to develop new mango varieties with pollen to withstand extreme weather. Temperature is a vital environmental factor that affects the plant reproductive process such as pollen germination, pollen tube growth and fruit setting and so on. This research results indicated that pollen germination rate and pollen tube length in vitro decreased seriously under high and low temperature stress, meanwhile the optimum temperature of four varieties were determined. Bilinear or modified bilinear regression models had been widely used to study the response of in vitro pollen germination to temperature and to screen heat-tolerant crops 35 . The observed pollen germination rates for cotton (44%) 18 , peanut (56%) 19 and coconut (40%) 36 under artificial medium were similar to the results of current study. Therefore, the response of pollen germination in vitro to temperature stress can be used as an accurate method to screen the best mango genotype in different ecological zones.
Among different mango varieties, the optimum temperature of pollen germination was significantly related with its maximum temperature. The highest optimum temperature of 'Renong No. 1' (30.4 ℃) also had the highest pollen germination rate (35.5%). Similar pollen condition occurred in maize 37 and Cucumis melo 38 . Meanwhile, among the four mango varieties, 'Renong No. 1' had the widest temperature range (T max − T min = 11.6 ℃), followed by 'Jinhuang' (11.5 ℃). In the commercial varieties promoted in Southern China, 'Renong No. 1' had the largest temperature range and wider temperature adaptability, while ' Apple mango' had the smallest temperature range (10.1 ℃) and weaker temperature adaptability during flowering period. It was consistent The optimum temperature of 'Jinhuang' was high, which was mainly planted in south subtropical region of Guizhou and Yunnan 41 ; The temperature adaptability of ' Apple mango' was weak, so it cannot be widely used as the main variety in southern China; The optimum temperature of 'Deshehari' was high, but it was sensitive to temperature change. It was mainly planted in Zhanjiang city, Guangdong province and the yield was unstable due to interannual climate change.
The basic temperature of mango pollen germination was 21.8 ℃ (T min ), 29.0 ℃ (T opt ) and 32.8 ℃ (T max ), respectively, it was similar to the cotton temperature of 14.0 ℃ (T min ), 31.0 ℃ (T opt ) and 43 ℃ (T max ) 19 as well as serpent cucumber 10 ℃ (T min ), 30 ℃ (T opt ) and 48 ℃ (T max ) 38 . Meanwhile, among the four promoted mango varieties in different provinces in southern China, 'Dashehari' had the narrowest pollen tube growth temperature range and the shortest growth length at the optimum temperature, which largely affected pollination and fertilization during flowering, and subsequent fruit setting and yield, the current result was confirmed by 'Dashehari' that was effected by weather and had unstable yield 42 . Similar to the response of pollen germination rate to temperature stress, the modified bilinear model described the response of pollen tube length under temperature stress. At the optimum temperature, the maximum length of pollen tube was from 51.2 μm of 'Dashehari' to 56.6 μm of 'Jinhuang' , the average value of 53.7 μm. The difference and range of pollen tube length observed in current study were similar to ornamental pepper 43 and soybean 44 and other crops cultured in artificial pollen germination solution. In the results of Brassica napus, Young et al. 45 pointed out that at high temperature, the poor fertility of pollen would lead to the decrease of pollen germination rate, which was the result of the loss of pollen moisture 46 ; Pressman et al. 47 suggested that high temperature affected the starch accumulation during pollen grains developed, which led to the decrease of the concentration of soluble sugar in mature pollen, so as to further reduce the pollen germination rate.
As for the adverse effects of temperature change on pollen germination, some researchers linked the response degree of different genotypes and even different species 48,49 , and tested the performance of pollen under different temperature stress 20,48 . Mango, widely cultivated in Southern China, often suffered from low temperature and www.nature.com/scientificreports/ rainy weather during flowering period in production, which led to abnormal meiosis of pollen mother cells, resulting in pollen fertilization and fruit drop 50 . Therefore, according to pollen germination rate and tube length CSRI value of different varieties of mango under different temperature stress, it was found that low temperature stress had a significant inhibitory effect on the growth of pollen tube, which was consistent to previous study 51 .
On the contrary, high temperature stress had a greater destructive effect on mango pollen germination than that of low temperature by analyzing the changes of pollen germination rate of four mango varieties under different temperature stress 52 . In order to compare the oxidative stress levels of different temperatures stresses on different genotypes of mango, the changes of enzymatic antioxidant activities and MDA content were measured. It is well known that enzymatic antioxidants such as SOD and POD can detoxify the excessive accumulation of ROS in pollen tubes caused by toxic stress [53][54][55] . In addition, when the content of ROS exceeds the threshold, lipid peroxidation occurs in both cell and organelle membrane, which had an adverse effect on normal cell function. MDA level was the final product of lipid peroxidation, which was often used as an indicator of ROS mediated cell membrane damage under temperature stress 32 . Gao et al. 56 discussed that under temperature stress induction, a large increase in MDA content was also detected in the pollen tube of Pyrus pyrifolia. The effect of high temperature on enzyme activity and MDA content was greater than that of low temperature on mango genotype. ' Apple mango' had the largest change rate of SOD, POD activity and MDA content under low temperature and high temperature conditions. At the same time, considering CSRI value and other factors, it can be seen that ' Apple mango' was the most sensitive to low temperature and high temperature stress. In addition, 'Renong No. 1' had the smallest change rate of SOD, POD activity and MDA content under low temperature and high temperature conditions. Therefore, 'Renong No. 1' was the genotype with the strongest resistance to low temperature and high temperature stress.
At present, with the occurrence of more and more extreme climates around the world, the damage of extreme weather to the plant production process was more serious compared to the rise of the average temperature in the whole mango planting season 57 . Therefore, it was vitally important to identify and cultivate mango varieties those can tolerant extreme weather change 58 .
Current research collected mango pollen during mango blooming period, four varieties were treated with different temperature stress, and estimated the maximum values of pollen germination rate (PG), pollen tube length (PTL) and their basic temperature (T min , T opt and T max ) according to quadratic equation and modified bilinear model. These effects of temperature on the germination characteristics of mango pollen were explored by using cumulative stress response index (CSRI), POD, SOD and MDA, as well as determination on the differences of mango varieties.
In all four mango genotypes, pollen tubes were more tolerant to low temperatures stress than that to high temperatures stress. Among the four genotypes, the ' Apple mango' genotype was the most sensitive to low temperature and high temperature stress, and the 'Renong No. 1' genotype was the most resistant to low temperature and high temperature stress. In conclusion, different temperatures had great effects on the flowering, pollination, fertilization and fruit setting of mango of the same genotype, and there were also significant differences between different genotypes. Current research results were of great significance for the introduction of new mango varieties in different ecological regions, the cultivation and management of mango at the flowering stage and the breeding of new mango varieties.

Materials and methods
Pollen collection. Mango pollen was collected from mango field genebank, South Subtropical Crops Research Institute, Chinese Academy of Tropical Agricultural Sciences in March 2021 (Zhanjiang, China). In the blooming period (15-75% of the flowering number in total flowers), mango trees of each varieties were randomly selected. The fresh anthers that bloomed but pollen not scattered on the same day were put into the culture solution, shaken sufficiently and quickly brought back to the laboratory for observation and analysis under different temperature stress.

Pollen germination and pollen tube length in vitro.
(1) Preparation of mango pollen culture solution: Mixed sucrose 150 g, boric acid 150 mg, calcium sulfate 6.0 mg, magnesium sulfate 100 mg, potassium nitrate 100 mg and polyethylene glycol 250 g, and dissolve to 1 L, which was heated and boiled to dissolve, then stored at normal temperature after cooled until used. (2) Pollen collection and pretreatment: the prepared culture solution was packed into 500 ul centrifuge tubes, from which picked 12-15 anthers randomly and fully vibrated to adhere the pollen to the culture solution. www.nature.com/scientificreports/ In the formula: Q is the germination rate; w is the number of germinated pollen; W is the total number of pollen; (5) For the length of pollen tube, three glass substrates were prepared, on which the length of about 50 germinated powder grains was measured by using optical microscope (Olympus bx-51).

Curve fitting equation.
In order to analyze the characteristics of pollen germination after temperature stress, linear and nonlinear regression models of temperature, maximum pollen germination rate and pollen tube length were established. In terms of R 2 and contribution rate of root mean square deviation (RMSD) of observation and fitted values, the fitting results of linear and nonlinear regression models were compared. The pollen germination rate and pollen tube length were obtained according to the quadratic linear model and the modified bilinear model, as well as the basic temperature of all genotypes based on the fitting equation.
The parameters of quadratic equation and modified bilinear equation are estimated according to nonlinear regression program PROC NLIN 59 . In terms of quadratic model, the minimum temperature ( T min ), the optimum temperature ( T opt )) and the maximum temperature ( T max ) were estimated by equation. The formula for calculating the pollen germination rate and basic temperature was as follows Eqs. (1)-(4): In this formula, PG was the pollen germination rate, T was the actual temperature, a,b,c are the genotype specific constants generated by PROC NLIN in SAS.
In terms of the modified bilinear equation of pollen tube growth and basic temperature, the following formula was shown in Eqs. (5)-(7): Of them,T ′ opt was fitted by SAS, T ′ min , T ′ max were determined by Eqs. (6) and (7).
Cumulative stress response index (CSRI). Taken pollen tube germination rate (GR) and pollen tube length (TL) of control group(c) and treatment croup (T) as indexes, the response of pollen tube to temperature stress was determined. The CSRI value was calculated using the following formula.

Determination of SOD and POD enzyme activity. SOD and POD enzyme reagent kit purchased from
Suzhou Grace Biotechnology Co., Ltd. (Suzhou, China), measurement of SOD and POD enzyme activity according to the manufacturer's manual. SOD activity is often determined by riboflavin NBT method. The enzyme activity was determined according to the inhibition of superoxide dismutase on the reduction of nitroblue tetrazole (NBT) under light. The amount of enzyme required to inhibit the photochemical reduction of NBT by 50% is one enzyme activity unit (U) 53,60 . POD activity is often determined by guaiacol method 61 . Peroxidase activity was determined by measuring the absorbance change at 470 nm wavelength. In the calculation of enzyme activity, the increase of absorbance A470 value per minute by 0.1 is one enzyme activity unit (U).
Determination of MDA content. MDA content was detected according to the method 62 . Weighed 0.5 g mango pollen, added 2 ml 5% TCA and a small amount of quartz sand, grind to the homogenate, added 8 ml TCA for further grinding, centrifuge the homogenate at 4000 r/min for 10 min, and the supernatant was the sample extract. Sucked 2 ml of supernatant, added 2 ml of 0.67% TBA solution and shake well. Put the test tube into boiling water bath and boiled for 10 min, took out the test tube and cooled it, and centrifuged for 15 min at 3000 r/min. The absorbance values at 532, 600 and 450 nm were measured with 0.67% TBA solution as blank.
(1) PG = a + bT − cT 2 www.nature.com/scientificreports/ Data analysis. Excel 2019 and SPSS 26.0 version software was used for statistical analysis. The difference was compared by using ANOVA with a threshold P value of 0.05.

Statement of research involving plants.
The collection of plant material and all methods were carried out in accordance with relevant guidelines of National Field Genebank for Tropical Fruit (Zhanjiang, China).

Data availability
The data that support the findings of this study are available from the corresponding author on reasonable request.